The brain microvasculature is comprised of a specialized class of endothelium that forms a cellular barrier between the bloodstream and the interstices of the brain. This so-called blood-brain barrier (BBB) distinguishes the brain microvasculature from peripheral vascular beds because it constitutes a physical and metabolic barrier that tightly regulates brain uptake of ions, small molecules, proteins, and circulating cells. Since brain endothelial cell plasma membranes contact both the bloodstream and brain interstitial fluid, they are ideally positioned to act as the controlling interfaces for signaling, immune regulation, and transport between the blood and brain. Therefore, many of the unique characteristics of the BBB endothelium can likely be attributed to the protein composition of its plasma membranes. In particular, the receptor-mediated transport (RMT) systems at the BBB play major functional roles in healthy brain function by controlling the transport of large molecules into and out of the brain. Moreover, RMT systems can also affect disease processes by either their dysfunction or by their direct participation in transporting substances involved in disease progression (e.g. beta-amyloid, HIV virus, measles virus). Finally, given the capability for RMT systems to transport large molecules, they have been increasingly used as a means for shuttling drug cargo into the brain noninvasively. Although RMT systems are a highly significant class of proteins, they have been understudied as a consequence of technological shortcomings. RMT receptors are membrane proteins that because of their low abundance and hydrophobicity are not well profiled using traditional two dimensional gel or tandem mass spectrometry methods. In addition, since the BBB comprises only 1/1000 of the brain total volume, proteomics studies using total brain tissue likely miss many of the important RMT systems in play at the BBB. Therefore, in this EUREKA proposal we will use a new, state-of-the-art technological platform developed in our laboratory that combines the power of antibody libraries with detergent-solubilized BBB plasma membrane lysates to simultaneously identify antibodies and cognate BBB RMT proteins. Because detergents can be employed, the hydrophobic nature of membrane proteins that often hampers other proteomics technologies can be overcome, thereby promising a broader coverage of the BBB RMT proteome (RMT-ome). Moreover, the approach enables such subcellular BBB membrane proteomics with high in vivo relevance because freshly isolated and subfractionated BBB is used as source tissue. Finally, our new approaches allow functional membrane proteomics in that identified membrane proteins can be readily categorized by their functional association with a generalized component of RMT systems. Taking advantage of these new tools, receptors and machinery that are involved in receptor-mediated transport (RMT) function at the BBB will be identified. Identified antibodies targeting RMT machinery will then used to assess RMT expression levels and tissue localization as well as to validate the transport function of the RMT machinery. Successful completion of the proposed research is predicted to have substantial impact not only for neuroscientists and cell biologists alike, but ultimately for the treatment of millions suffering from neurological disease.